The long-term goal of this research is to investigate the role of the alternative sigma factors SigD, SigE, SigH and SigM in the regulation of Mycobacterium tuberculosis gene expression and virulence. The underlying hypothesis of this research is that extracytoplasmic function (ECF) sigma factors of M tuberculosis play an important role in the regulation of gene expression during the interaction of this organism with the host during infection, and that this regulation is important for the virulence of this organism. This proposal has four complementary specific aims. The first aim is to complete construction of sigma factor mutant strains of M tuberculosis and to compare the survival of these mutants vs. the parental strain H37Rv following in vitro oxidative and nitrosative stresses that may be relevant to infection. The second aim is to compare the virulence of these mutants vs. H37Rv in macrophage and mouse models of infection. Mutant strains will be complemented to verify that any phenotypes observed in these experiments result from the disruption of the sigma factor gene that was mutated. The third aim is to investigate the role of these sigma factors in regulating M tuberculosis gene expression, focusing on the use of microarray technology, supplemented with bioinformatic and traditional molecular genetic methods. The fourth aim is to investigate the role of specific sigma factor-regulated genes in M tuberculosis virulence, by examining their expression and by constructing and testing M tuberculosis strains with mutations in these genes. These investigations are expected to identify regulatory networks and specific genes that are important in M tuberculosis virulence. This research thus has the potential to generate new insights into the pathogenesis of M tuberculosis infection, and may provide the basis for new approaches to the control of tuberculosis.